- Email: [email protected]
657. MicroRNA-Based Small Interfering RNA Silencing for Inhibiting Apoptosis
Oligonucleotide Therapies I N=5) administered intracheally on day 1 and 3 with the aerosolised nanoparticles (0.26 µg per dose). Flow cytometric analysis of digested lung tissue showed significant EGFP knockdown characterised by lower EGFP fluorescence ratio (defined as ratio of EGFP positive to EGFP negative cells) in the EGFP-specific nanoparticle formulation (~10 ratio) compared to non-formulated EGFP (~45 ratio) or mismatch formulated controls (~30 ratio). This work provides a platform for more effective pulmonary delivery and gene silencing of RNAi therapeutics that may be applied in the treatment of respiratory diseases.
655. Intracellular Time-Controlled Delivery of siRNA from Cationic Nanogel Depots Koen Raemdonck, Joseph Demeester, Stefaan C. De Smedt. 1 Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Ghent, Belgium. 1
1
1
Since the demonstration of RNA interference (RNAi) in mammalian cells, considerable research and financial effort has gone to the implementation of RNAi as a viable therapeutic platform for the treatment of human genetic disorders. To apply siRNA as a therapeutic, both the intensity and duration of the gene silencing effect will have to be controlled and optimized according to the selected disease target. We anticipate that an intracellular time-controlled release of siRNA could be an interesting approach to maintain the cytosolic siRNA concentration above a critical threshold for a longer period of time. The packaging of siRNA in non-viral carriers that shield the siRNA from its direct environment and slowly releases it into the cytosol could be advantageous to prolong their silencing effect. Moreover, adverse effects originating from saturation of the RNAi pathway or unwanted immune activation may be minimized when obtaining better control over the intracellular siRNA concentrations. To achieve such goals it is evident that carriers with flexible siRNA release properties are needed. In this abstract, two types of cationic biodegradable dextran nanogels will be discussed for the intracellular time-controlled release of siRNA. A first preparation method comprises the application of lipid vesicles as nanoreactors for the selective photopolymerization of hydrolysable methacrylated dextran in their aqueous lumen. In this way lipid-coated dextran nanogels are prepared that combine the properties of liposomes and biodegradable nanoparticles. Secondly, dextran nanogels may also be produced through mini-emulsion photopolymerization. These naked (i.e. non-lipid coated) cationic nanogels are able to absorbe and encapsulate siRNA based on electrostatic attraction. Irrespective of the electrostatic nature of the siRNA incorporation, still a controlled siRNA release can be achieved. Data obtained on microgels showed that, depending on the hydrogel network properties, the siRNA release profile can be tailored from hours to days. A broad spectrum of cationic gels can be prepared by varying the crosslink density of the methacrylated dextran and the type of cationic methacrylate monomer used for copolymerization. Both lipid-coated and naked nanogels can easily be taken up by hepatoma cells. Cationic nanogels, loaded with siRNA, are able to silence the expression of luciferase in hepatoma cells that are stably transfected with the firefly luciferase gene. The silencing efficiency of the nanogels can be enhanced by stimulating endosomal escape of the nanocomplex. Once inside the cytosol, the nanogels function as a siRNA depot, slowly disintegrating through network hydrolysis thereby releasing the entrapped siRNA drugs.
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
656. Small Fragment Homologous Replacement (SFHR) Correction of HPRT Mutations in Hydorxyurea Treated Lymphoblastoid Cells
Hooman Parsi,1 Babak Bedayat,1 Hamid Emamekhoo,1,4 Janice A. Nicklas,2 Dieter C. Gruenert.1,3,4 1 Department of Cell Biology, California Pacific Medical Center Research Institute, San Francisco, CA; 2Department of Pediatrics, University of Vermont, Burlington, VT; 3Department of Medicine, University of Vermont, Burlington, VT; 4Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA. Gene therapy with oligonucleotide-based, sequence-specific modification methods offers potentially safe and effective alternatives to traditional cDNA-based approaches. One of oligonucleotide-based gene targeting methods, SFHR was used to modify a single base mutation in exon 3 of the X-chromosome linked human hypoxanthineguanine phosphoribosyl transferase (HPRT) gene. This point mutation inactivates the HPRT gene and renders the cells unable to grow in hypoxanthine/aminopterin/thymidine (HAT) containing medium. When male lymphoblastoid cells were transfected with small DNA fragments (SDFs) in the presence of hydroxyurea (HU) there was an apparent enhancement of SFHR-mediated correction of the target locus. Cells were incubated for 24h and 48h in the presence of 1mM HU and then transfected by the Amaxa nucleofection system with a 579-bp wild type (wt)SDF compromising exon 3 of HPRT. One week post-transfection the cells incubated for 24h prior to transfection showed normal growth, while cells preincubated for 48h showed no growth. The growing cells were then placed into HAT medium for selection of HAT resistance and normal HPRT function. Genomic DNA and RNA of cells growing in HAT were isolated and analyzed by PCR and RT-PCR, respectively. Cells that were transfected with 107 double stranded SDF per cell not only grew in HAT, but showed sequence-specific correction of the DNA and RNA. Preincubation of the cells with 10 µg/ml aphidicolin, a concentration that is generally used to inhibit DNA synthesis, was toxic to the cells. It is possible that a transient block in S phase DNA replication induced by HU can further increase the efficiency of SFHR and decrease the amount of SDF required for modification.
657. MicroRNA-Based Small Interfering RNA Silencing for Inhibiting Apoptosis
Guofeng Cheng,1 Ram I. Mahato.1 1 Department of Pharmaceutical Sciences, University of Tennessee Health Sciences Center, Memphis, TN. Introduction. Pancreatic islet transplantation has the potential to become an effective method for treating type I diabetes. However, most grafted islets fail to function due to host immune rejection, nonspecific inflammatory response and poor revascularization. Since caspase-3 plays a crucial role in apoptosis of transplanted islets, we used small interfering RNAs (siRNAs) to silence caspase-3 in insulinoma (INS-1E) cells and human islets and determine whether antiapoptic gene silencing can improve the outcome of islet transplantation. Methods. Three different siRNA duplexes targeting rat caspase-3 and one validated siRNA targeting human caspase-3 were chemical synthesized and transfected into INS-1E cells and human islets after complex formation with Lipofectamine 2000. Then short hairpin RNAs (shRNAs) were designed based on the most potent siRNA sequence for cloning into expressing vector (pSilencer 1.0) and adenoviral vectors. Results. The level of caspase-3 transcripts were reduced by about 50-67% in INS-1E cells and 50% in human islets upon transfection of siRNAs. Apoptosis in beta cells was markedly inhibited as determined by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL). Transfection process did not affect the insulin secretion capability of the islets. Introduction S245
Oligonucleotide Therapies I of microRNA375-based shRNA in INS-1E cells resulted in 50% reduction of caspase-3 at the transcript level and 20% reduction at protein level at 72 hours post transfection. Transduction of human islets with adenoviral vector encoding shRNA reduced caspase-3 mRNA by 70% and protein levels by 60% at day 5 as compared with those at day 3. However, this effect was not observed with control adenoviral transduction. Conclusions. We demonstrated 1) caspase-3 gene silencing can significantly prevent insulinoma cells from apoptosis and does not affect islet function, 2) microRNA375based shRNA can further facilitate this silencing effect, and 3) the recombinant adenovirus encoding shRNA offers relatively longterm and effective silencing effect in human islets. In conclusion, our observations suggest that siRNA/shRNA targeting caspase-3 may be of therapeutic value and may improve the outcome of islet transplantation. Acknowledgements The study was support by the NIH (1R01DK69968). References Emamaullee, J.A., et al. (2007) Diabetes. 56:1289-1298.
658. The Primary MicroRNA Background Affects the Targeting Activity of Different Pol II-Expressed Anti-HIV Guide Sequences Samantha Barichievy,1 Sheena Saayman,1 Marc S. Weinberg.1 1 Department of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, Gauteng, South Africa.
Silencing of HIV-1 by RNA Interference (RNAi) has been achieved using short hairpin RNAs (shRNAs), which are expressed from RNA Pol III cassettes, and are processed by the RNAse III enzyme Dicer to generate active siRNAs. However, to prevent viral escape, a combination of more than one antiviral RNAi effector sequence is required. Multiple RNA Pol III expression cassettes have been developed, but these may result in saturation of the endogenous RNAi pathway and offer no possibility for localized control in gene expression. Polycistronic primary microRNAs (pri-miRs) have the advantage of being expressed from a single RNA Pol II transcript and target multiple sequences simultaneously. This study aimed to characterise the role pri-miRs with different anti-HIV effector sequences as viable suppressors of HIV-1 targets. Four previously characterized anti-HIV RNAi effector sequences targeting conserved sites within LTR, GAG, INT and TAT were chosen for further study. All four effector sequences were included into expression cassettes encoding pri-miRNA mimicked precursors for pri-miR-30a, primiR-31, pri-miR-122 and pri-miR-155. The inhibitory efficacy of each pri-miR cassette was determined in transient transfection experiments against a pNL4-3/Luc HIV-1 luciferase reporter gene target. Different vector to target ratios were used and compared to respective Pol III-expressing shRNA controls. Even though all primiR mimics were capable of significant knockdown in HEK293 and Huh-7 cells, pri-miR-30a and pri-miR-155 were particularly effective with up to 95% target inhibition. Pri-miR-122 mimics were the least effective. Results with different effector sequences were uniform for each pri-miR mimic except for pri-miR-31, which displayed a large range of inhibitory effects between anti-HIV effector sequences. Northern blot analysis of guide strand RNA for each target indicated that Drosha/Dicer recognition and processing is probably the most important determinant for effective pri-miR mimic construction. An important future objective remains the construction of a transcript containing polycistronic pri-miRNA mimics to generate up to four anti-HIV effector sequences. The results obtained in this study provide some guiding principles for the effectiveness of different pri-miR mimics currently in use for suppressive gene therapy approaches.
S246
659. Skin Application of Chitosan – Antisense Oligonucleotides Complexes
Ali Demir Sezer, Suna Ozbas-Turan, Julide Akbuga. 1 Pharmaceutical Biotechnology, Faculty of Pharmacy, Marmara University, Istanbul, Turkey. Skin delivery of antisense oligonucleotides (As ODN) has exciting potential in the treatment of skin diseases. However, the therapeutic applications of oligonucleotide-based therapies are limited by the instability of these molecules towards nucleases, short half-life in vivo and insufficient cellular update. Thus the use of nonviral carriers such as chitosan may be more realistic approach to deliver As ODN. Chitosan is an attractive vector for gene delivery because of its high positive charge density, relatively low toxicity and high transfection efficiency. The aim of this study is to investigate skin delivery potential of chitosan / As ODN complexes in rats. 15-nucleotide phosphorothioate oligonucleotide (MWG-Biotech Germany) designed to target β-gal gene and chitosan (400 kD, Fluka) were used complexes were prepared according to our earlier report [1]. Chitosan – As ODN complexes (50:1) were applied to Sprague Dowley rats (8 weeks old). Complexes [500 µl PBS (pH 7.4) containing 15µg As ODN] were spread on the shave area (hairless) at the back of the animals (n=6) after pSV-β-gal plasmid application. After 24 hours of applications, animals were scarified and skin samples were taken for measurement. β-gal expression was spectrophotometrically measured in the samples with ONPG. X-gal staining was applied on samples for histological control. Total protein concentration was assayed according to Bradford’s method. Untreated group of animals was used as a control. After topical application of the complexes in different doses (7.5, 15 and 30 µg As ODN), β-gal expression reduced significantly (P<0.001). Depend on the dose used, percent inhibition of β-gal expression changed between 59.95% - 90.11%. The highest gene inhibition (90.11%) were obtained after the applications of 30µg As ODN to the rats (P<0.001). In conclusion, chitosan may be an attractive carrier for skin delivery of antisense oligonucleotides. References [1]. Enneli, B., E. Salva., Akbuga, J. Development of a novel delivery system for antisense oligonucleotides. FEBS Journal. 273 (2006) 132. Acknowledgements This study was supported by Commission of Marmara University Scientific Research Project (BAPKO, SAG-BGS-120707-0143).
660. Efficient Inhibition of Hepatitis C Virus Replication by Oligodeoxyribozymes That Cleave Viral Genome RNA Ki-Sun Kim,1 Bok Hee Lee,1 Joon-Sik Choi,2 Dong-Eun Kim.1 Bioscience and Biotechnology, Konkuk Univesity, Seoul, Korea; 2 Biochemistry, Chungnam National University, Daejeon, Korea.
1
A class of antisense oligodeoxyribozymes, known as the 10-23 DNAzyme, has been shown to efficiently cleave target RNA at purine-pyrimidine junctions in vitro. We have utilized a strategy to identify accessible cleavage sites for DNAzyme in the target RNA, the hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool of random DNAzyme library. The screening procedure identified 18 potential cleavage sites in the target RNA. Corresponding DNAzymes were constructed for the selected target sites and were tested for RNA-cleavage in vitro. The selected DNAzymes (Dz#4 and Dz#6), when transfected to the human hepatoma cells harboring the HCV subgenomic replicon RNA, efficiently inhibited HCV RNA replication in cells by reducing expression of HCV NS3 RNA and protein. In addition, shRNA was also constructed against the same targeting region with the Dz#6 to induce RNA interference in HCV genomic RNA expression. Both DNAzyme and shRNA was effective in inhibiting the replication of HCV RNA genome in the cells. Thus, the selected oligonucleotides as well as the selection strategy can be applied for development of new Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
655. Intracellular Time-Controlled Delivery of siRNA from Cationic Nanogel Depots Koen Raemdonck, Joseph Demeester, Stefaan C. De Smedt. 1 Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Ghent, Belgium. 1
1
1
Since the demonstration of RNA interference (RNAi) in mammalian cells, considerable research and financial effort has gone to the implementation of RNAi as a viable therapeutic platform for the treatment of human genetic disorders. To apply siRNA as a therapeutic, both the intensity and duration of the gene silencing effect will have to be controlled and optimized according to the selected disease target. We anticipate that an intracellular time-controlled release of siRNA could be an interesting approach to maintain the cytosolic siRNA concentration above a critical threshold for a longer period of time. The packaging of siRNA in non-viral carriers that shield the siRNA from its direct environment and slowly releases it into the cytosol could be advantageous to prolong their silencing effect. Moreover, adverse effects originating from saturation of the RNAi pathway or unwanted immune activation may be minimized when obtaining better control over the intracellular siRNA concentrations. To achieve such goals it is evident that carriers with flexible siRNA release properties are needed. In this abstract, two types of cationic biodegradable dextran nanogels will be discussed for the intracellular time-controlled release of siRNA. A first preparation method comprises the application of lipid vesicles as nanoreactors for the selective photopolymerization of hydrolysable methacrylated dextran in their aqueous lumen. In this way lipid-coated dextran nanogels are prepared that combine the properties of liposomes and biodegradable nanoparticles. Secondly, dextran nanogels may also be produced through mini-emulsion photopolymerization. These naked (i.e. non-lipid coated) cationic nanogels are able to absorbe and encapsulate siRNA based on electrostatic attraction. Irrespective of the electrostatic nature of the siRNA incorporation, still a controlled siRNA release can be achieved. Data obtained on microgels showed that, depending on the hydrogel network properties, the siRNA release profile can be tailored from hours to days. A broad spectrum of cationic gels can be prepared by varying the crosslink density of the methacrylated dextran and the type of cationic methacrylate monomer used for copolymerization. Both lipid-coated and naked nanogels can easily be taken up by hepatoma cells. Cationic nanogels, loaded with siRNA, are able to silence the expression of luciferase in hepatoma cells that are stably transfected with the firefly luciferase gene. The silencing efficiency of the nanogels can be enhanced by stimulating endosomal escape of the nanocomplex. Once inside the cytosol, the nanogels function as a siRNA depot, slowly disintegrating through network hydrolysis thereby releasing the entrapped siRNA drugs.
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
656. Small Fragment Homologous Replacement (SFHR) Correction of HPRT Mutations in Hydorxyurea Treated Lymphoblastoid Cells
Hooman Parsi,1 Babak Bedayat,1 Hamid Emamekhoo,1,4 Janice A. Nicklas,2 Dieter C. Gruenert.1,3,4 1 Department of Cell Biology, California Pacific Medical Center Research Institute, San Francisco, CA; 2Department of Pediatrics, University of Vermont, Burlington, VT; 3Department of Medicine, University of Vermont, Burlington, VT; 4Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA. Gene therapy with oligonucleotide-based, sequence-specific modification methods offers potentially safe and effective alternatives to traditional cDNA-based approaches. One of oligonucleotide-based gene targeting methods, SFHR was used to modify a single base mutation in exon 3 of the X-chromosome linked human hypoxanthineguanine phosphoribosyl transferase (HPRT) gene. This point mutation inactivates the HPRT gene and renders the cells unable to grow in hypoxanthine/aminopterin/thymidine (HAT) containing medium. When male lymphoblastoid cells were transfected with small DNA fragments (SDFs) in the presence of hydroxyurea (HU) there was an apparent enhancement of SFHR-mediated correction of the target locus. Cells were incubated for 24h and 48h in the presence of 1mM HU and then transfected by the Amaxa nucleofection system with a 579-bp wild type (wt)SDF compromising exon 3 of HPRT. One week post-transfection the cells incubated for 24h prior to transfection showed normal growth, while cells preincubated for 48h showed no growth. The growing cells were then placed into HAT medium for selection of HAT resistance and normal HPRT function. Genomic DNA and RNA of cells growing in HAT were isolated and analyzed by PCR and RT-PCR, respectively. Cells that were transfected with 107 double stranded SDF per cell not only grew in HAT, but showed sequence-specific correction of the DNA and RNA. Preincubation of the cells with 10 µg/ml aphidicolin, a concentration that is generally used to inhibit DNA synthesis, was toxic to the cells. It is possible that a transient block in S phase DNA replication induced by HU can further increase the efficiency of SFHR and decrease the amount of SDF required for modification.
657. MicroRNA-Based Small Interfering RNA Silencing for Inhibiting Apoptosis
Guofeng Cheng,1 Ram I. Mahato.1 1 Department of Pharmaceutical Sciences, University of Tennessee Health Sciences Center, Memphis, TN. Introduction. Pancreatic islet transplantation has the potential to become an effective method for treating type I diabetes. However, most grafted islets fail to function due to host immune rejection, nonspecific inflammatory response and poor revascularization. Since caspase-3 plays a crucial role in apoptosis of transplanted islets, we used small interfering RNAs (siRNAs) to silence caspase-3 in insulinoma (INS-1E) cells and human islets and determine whether antiapoptic gene silencing can improve the outcome of islet transplantation. Methods. Three different siRNA duplexes targeting rat caspase-3 and one validated siRNA targeting human caspase-3 were chemical synthesized and transfected into INS-1E cells and human islets after complex formation with Lipofectamine 2000. Then short hairpin RNAs (shRNAs) were designed based on the most potent siRNA sequence for cloning into expressing vector (pSilencer 1.0) and adenoviral vectors. Results. The level of caspase-3 transcripts were reduced by about 50-67% in INS-1E cells and 50% in human islets upon transfection of siRNAs. Apoptosis in beta cells was markedly inhibited as determined by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL). Transfection process did not affect the insulin secretion capability of the islets. Introduction S245
Oligonucleotide Therapies I of microRNA375-based shRNA in INS-1E cells resulted in 50% reduction of caspase-3 at the transcript level and 20% reduction at protein level at 72 hours post transfection. Transduction of human islets with adenoviral vector encoding shRNA reduced caspase-3 mRNA by 70% and protein levels by 60% at day 5 as compared with those at day 3. However, this effect was not observed with control adenoviral transduction. Conclusions. We demonstrated 1) caspase-3 gene silencing can significantly prevent insulinoma cells from apoptosis and does not affect islet function, 2) microRNA375based shRNA can further facilitate this silencing effect, and 3) the recombinant adenovirus encoding shRNA offers relatively longterm and effective silencing effect in human islets. In conclusion, our observations suggest that siRNA/shRNA targeting caspase-3 may be of therapeutic value and may improve the outcome of islet transplantation. Acknowledgements The study was support by the NIH (1R01DK69968). References Emamaullee, J.A., et al. (2007) Diabetes. 56:1289-1298.
658. The Primary MicroRNA Background Affects the Targeting Activity of Different Pol II-Expressed Anti-HIV Guide Sequences Samantha Barichievy,1 Sheena Saayman,1 Marc S. Weinberg.1 1 Department of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, Gauteng, South Africa.
Silencing of HIV-1 by RNA Interference (RNAi) has been achieved using short hairpin RNAs (shRNAs), which are expressed from RNA Pol III cassettes, and are processed by the RNAse III enzyme Dicer to generate active siRNAs. However, to prevent viral escape, a combination of more than one antiviral RNAi effector sequence is required. Multiple RNA Pol III expression cassettes have been developed, but these may result in saturation of the endogenous RNAi pathway and offer no possibility for localized control in gene expression. Polycistronic primary microRNAs (pri-miRs) have the advantage of being expressed from a single RNA Pol II transcript and target multiple sequences simultaneously. This study aimed to characterise the role pri-miRs with different anti-HIV effector sequences as viable suppressors of HIV-1 targets. Four previously characterized anti-HIV RNAi effector sequences targeting conserved sites within LTR, GAG, INT and TAT were chosen for further study. All four effector sequences were included into expression cassettes encoding pri-miRNA mimicked precursors for pri-miR-30a, primiR-31, pri-miR-122 and pri-miR-155. The inhibitory efficacy of each pri-miR cassette was determined in transient transfection experiments against a pNL4-3/Luc HIV-1 luciferase reporter gene target. Different vector to target ratios were used and compared to respective Pol III-expressing shRNA controls. Even though all primiR mimics were capable of significant knockdown in HEK293 and Huh-7 cells, pri-miR-30a and pri-miR-155 were particularly effective with up to 95% target inhibition. Pri-miR-122 mimics were the least effective. Results with different effector sequences were uniform for each pri-miR mimic except for pri-miR-31, which displayed a large range of inhibitory effects between anti-HIV effector sequences. Northern blot analysis of guide strand RNA for each target indicated that Drosha/Dicer recognition and processing is probably the most important determinant for effective pri-miR mimic construction. An important future objective remains the construction of a transcript containing polycistronic pri-miRNA mimics to generate up to four anti-HIV effector sequences. The results obtained in this study provide some guiding principles for the effectiveness of different pri-miR mimics currently in use for suppressive gene therapy approaches.
S246
659. Skin Application of Chitosan – Antisense Oligonucleotides Complexes
Ali Demir Sezer, Suna Ozbas-Turan, Julide Akbuga. 1 Pharmaceutical Biotechnology, Faculty of Pharmacy, Marmara University, Istanbul, Turkey. Skin delivery of antisense oligonucleotides (As ODN) has exciting potential in the treatment of skin diseases. However, the therapeutic applications of oligonucleotide-based therapies are limited by the instability of these molecules towards nucleases, short half-life in vivo and insufficient cellular update. Thus the use of nonviral carriers such as chitosan may be more realistic approach to deliver As ODN. Chitosan is an attractive vector for gene delivery because of its high positive charge density, relatively low toxicity and high transfection efficiency. The aim of this study is to investigate skin delivery potential of chitosan / As ODN complexes in rats. 15-nucleotide phosphorothioate oligonucleotide (MWG-Biotech Germany) designed to target β-gal gene and chitosan (400 kD, Fluka) were used complexes were prepared according to our earlier report [1]. Chitosan – As ODN complexes (50:1) were applied to Sprague Dowley rats (8 weeks old). Complexes [500 µl PBS (pH 7.4) containing 15µg As ODN] were spread on the shave area (hairless) at the back of the animals (n=6) after pSV-β-gal plasmid application. After 24 hours of applications, animals were scarified and skin samples were taken for measurement. β-gal expression was spectrophotometrically measured in the samples with ONPG. X-gal staining was applied on samples for histological control. Total protein concentration was assayed according to Bradford’s method. Untreated group of animals was used as a control. After topical application of the complexes in different doses (7.5, 15 and 30 µg As ODN), β-gal expression reduced significantly (P<0.001). Depend on the dose used, percent inhibition of β-gal expression changed between 59.95% - 90.11%. The highest gene inhibition (90.11%) were obtained after the applications of 30µg As ODN to the rats (P<0.001). In conclusion, chitosan may be an attractive carrier for skin delivery of antisense oligonucleotides. References [1]. Enneli, B., E. Salva., Akbuga, J. Development of a novel delivery system for antisense oligonucleotides. FEBS Journal. 273 (2006) 132. Acknowledgements This study was supported by Commission of Marmara University Scientific Research Project (BAPKO, SAG-BGS-120707-0143).
660. Efficient Inhibition of Hepatitis C Virus Replication by Oligodeoxyribozymes That Cleave Viral Genome RNA Ki-Sun Kim,1 Bok Hee Lee,1 Joon-Sik Choi,2 Dong-Eun Kim.1 Bioscience and Biotechnology, Konkuk Univesity, Seoul, Korea; 2 Biochemistry, Chungnam National University, Daejeon, Korea.
1
A class of antisense oligodeoxyribozymes, known as the 10-23 DNAzyme, has been shown to efficiently cleave target RNA at purine-pyrimidine junctions in vitro. We have utilized a strategy to identify accessible cleavage sites for DNAzyme in the target RNA, the hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool of random DNAzyme library. The screening procedure identified 18 potential cleavage sites in the target RNA. Corresponding DNAzymes were constructed for the selected target sites and were tested for RNA-cleavage in vitro. The selected DNAzymes (Dz#4 and Dz#6), when transfected to the human hepatoma cells harboring the HCV subgenomic replicon RNA, efficiently inhibited HCV RNA replication in cells by reducing expression of HCV NS3 RNA and protein. In addition, shRNA was also constructed against the same targeting region with the Dz#6 to induce RNA interference in HCV genomic RNA expression. Both DNAzyme and shRNA was effective in inhibiting the replication of HCV RNA genome in the cells. Thus, the selected oligonucleotides as well as the selection strategy can be applied for development of new Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy